Is the Combination of Platelet-Rich Plasma and Hyaluronic Acid the Best Injective Treatment for Grade II-III Knee Osteoarthritis? A Prospective Study

Background Knee osteoarthritis is a common disease with increasing incidence and prevalence in western countries. It can cause severe pain and functional limitations, thereby representing a threat for patients' quality of life and a burden for national health systems. Intra-articular injections with hyaluronic acid (HA) and platelet-rich plasma (PRP) have been used for decades in order to reduce the symptoms caused by osteoarthritis. In recent years, a combination of HA and PRP has been introduced in clinical practice with the aim to minimize the clinical presentation of osteoarthritis and potentially delay articular degeneration. Materials and Methods Sixty cases with grade II-III knee osteoarthritis according to the Kellgren–Lawrence classification were included in a prospective study, focused on the evaluation of clinical and functional outcomes after intra-articular knee injections. Cases were randomly divided into three groups. Twenty cases (Group A) were injected with HA, 20 (Group B) had PRP, and the remaining 20 (Group C) received a combination of HA and PRP. Basal WOMAC score and VAS score were recorded before the treatment and repeated within 3 and 6 months after the treatment. Results At 6-month follow-up, Group C (PRP + HA) was the one with the lowest WOMAC and VAS mean values. It was also the only group that reported a reduction in the two values both in the first three months and in the following three months. No major complication was recorded. Conclusion The combination of platelet-rich plasma and hyaluronic acid can be effective in the treatment of grade II-III knee osteoarthritis in a short-to-mid-term scenario. It represents an innovative and valuable alternative to the administration of its two components alone.


Introduction
Osteoarthritis is the most common disease of joints in the adult population [1][2][3][4]. In particular, the knee is the most frequent location for osteoarthritis, as its prevalence in adults is around 6%, rising as high as 40% among the 70-to-74-year-olds [5][6][7]. Knee osteoarthritis thereby represents a burden for national health systems, as it can lead to serious disability caused by increasing pain and progressive articular stifness that may limit patients' autonomy and thereby have a negative impact on their quality of life [8][9][10][11][12]. Te therapeutic goal for those cases that sufer from mild-to-moderate knee osteoarthritis is to alleviate the signs and symptoms of the disease and, if possible, to slow its progression. In this scenario, intra-articular injections with antiinfammatory or viscosupplementation drugs represent a therapeutic option capable of reducing patients' pain and restore part of their previous functionality, as testifed by several meta-analyses [13,14] and randomized controlled trials [15,16].
Discovered in 1934 by Karl Meyer and John Palmer, the hyaluronic acid (HA) has been used since the 1970s to treat articular pain due to mild and moderate knee osteoarthritis. For almost half a century, HA has been one of the most used injective drugs for the treatment of osteoarthritis considering both its physical-chemical properties and its interaction with cartilages' cells and extracellular matrixes (ECMs) [17]. In the last decades, the continuous search for the discovery of new and efective injective treatments has driven the interest in regenerative biologic therapies [18]. Autologous platelet-rich plasma (PRP) has been introduced as an alternative to HA for cases sufering from moderate knee osteoarthritis, leading to encouraging results in terms of both pain relief and functional recovery [19,20]. A further innovation of the two approaches is represented by the administration of PRP alongside HA, introduced in recent years and performed with the theoretical aim to combine the efects of these two drugs in order to maximize and extend their efcacy. To this date, only a limited number of studies have evaluated the efectiveness of this combination despite some encouraging preliminary results [21][22][23][24][25]. Te aim of our study is to evaluate the efectiveness of the association of PRP and HA, comparing it with its two components administered alone. Te efectiveness of injective treatments was assessed through the months that followed their administration in a single institution on similar cohorts of patients sufering from comparable grades of knee osteoarthritis (grade II-III according to the Kellgren-Lawrence classifcation). Providing the best possible standardization between the three groups (HA, PRP, and PRP + HA), we investigated whether the combination of PRP and HA could be more efective than PRP or HA alone in terms of functional recovery and pain control on short-term and mid-term scenarios.

. Materials and Methods
Tis single-center double-blinded prospective study was approved by our Ethical Committee (CEAVNO CE150034-835/2015) and performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. All patients gave their written consent. Te study was conduced between January 2018 and January 2020.
Inclusion criteria for the inclusion in our research were a radiographic evidence of knee osteoarthritis (grade II-III according to the Kellgren-Lawrence classifcation), pain or functional limitations in patients' activity of daily living before the treatment, being between 30 and 80 years of age, and the absence of clinical or imaging signs of articular instability. Exclusion criteria were known hypersensitivity to hyaluronic acid, pregnancy and lactation, a BMI higher than 40, chronic administration of anticoagulant drugs or a history of coagulopathies, neoplastic lesions, and knee or kidney failures.
Sixty eligible cases were included in our study. Gender, age, and body mass index (BMI) of each patient were recorded at the moment of their inclusion in our study. Te clinical picture of each case was analyzed before the treatment using the Western Ontario McMaster Universities Osteoarthritis Index (WOMAC) to evaluate patients' overall clinical condition and the visual analogue scale (VAS) to assess their pain in particular.
Patients were randomly divided into three groups, with 20 subjects (10 males and 10 females) for each group. Group A was treated with intra-articular injections of hyaluronic acid (HA) (ArthroVisc; Regen Lab, Le Mont-sur-Lausanne, Switzerland), and Group B was treated with intra-articular autologous platelet-rich plasma (PRP) (RegenKit-BCT-1; Regen Lab, Le Mont-sur-Lausanne, Switzerland), whereas Group C received a combination of platelet-rich plasma and hyaluronic acid (Cellular Matrix A-CP-HA kit; Regen Lab, Le Mont-sur-Lausanne, Switzerland).
Te drug was administered in our clinic on a monthly basis, for a total of three intra-articular injections through two months. Te injections were performed in sterile conditions. Every eventual adverse reaction to the drugs was recorded and reported. Further outpatient visits were performed three and six months after the last injection, evaluating each patients' clinical outcomes in terms of VAS and WOMAC scores. Tese values, compared to the ones obtained before the treatment, were used to assess the efectiveness of the combination of PRP and HA compared to the two components administered alone.

Materials.
Te hyaluronic acid used in our study was ArthroVisc (Regen Lab, Le Mont-sur-Lausanne, Switzerland). Te product consists of a 2 ml syringe containing 40 mg of fermented hyaluronic acid with a molecular weight of 1550 kDa.
Te administration of PRP alone was performed using the RegenKit-BCT-1 (Regen Lab, Le Mont-sur-Lausanne, Switzerland). For each patient, 8 ml of peripheral circulating blood was collected in a proper tube and centrifuged at 1500g for fve minutes, obtaining 4 ml of platelet-rich plasma. Te PRP was then drawn into a sterile syringe and injected in the patient's knee.
Te injections of PRP alongside HA were performed using the Cellular Matrix A-CP-HA kit (Regen Lab, Le Mont-sur-Lausanne, Switzerland). Te kit was provided with tubes that allowed to mix 2 ml/40 mg of hyaluronic acid (1550 kDa) with 4 ml of platelet-rich plasma obtained with the aforementioned process. At the end of the preparation phase, a sterile syringe containing 4 ml of PRP and 2 ml of HA was injected in the target knee.
All the three kits used in our study were produced by the same company. Te chemical properties of the hyaluronic acid injected alone were the same as the one that was combined with PRP. Te tubes and the other devices of the kits used to prepare PRP and PRP + HA were comparable, and both kits were used with the same centrifugation system.

Statistics.
Statistical analysis was performed using Stata SE 13 (StataCorp LLC). Statistical signifcance was set at 0.05 for all endpoints. Descriptive analyses were performed for each value of VAS and WOMAC scores. T-student tests were used to assess eventual diferences between the results obtained by those who received PRP + HA and the ones obtained by other cohorts.
Cases belonging to Group A (HA) had a mean basal VAS score of 5.5, a value that decreased to 4.3 after three months and 4.0 after six months (Figure 1). Teir mean basal WOMAC score was 36.4 at base time, 28.8 after three months, and 31.8 after six months ( Figure 2). Cases of Group B (PRP) had a mean VAS score of 6.1 before injections, 3.1 after three months, and 3.5 after six months (Figure 1). Teir WOMAC score was 41.5 at the beginning of our study and 19.6 after three months, remaining steady three months later ( Figure 2).
Cases in Group C (PRP + HA) saw a distinct reduction of their mean VAS score through our study, moving from a starting value of 5.2 to 2.9 after three months to 2.4 after six months ( Figure 1). Te mean WOMAC score decreased from 33.7 to 21.4 at three-month follow-up and later to 17.5 at fnal six-month follow-up ( Figure 2).
Te aforementioned results are summarized in Table 1.
Te mean diferential VAS score between the starting value and the one recorded six months after the end of the treatment was 1.6 in Group A, 2.6 in Group B, and 2.8 in Group C (Figure 1).
One-tailed t-student tests on VAS scores taken six months after treatments testifed a signifcantly better pain relief in Group C compared to both Group A and Group B (p � 0.005, p � 0.043). Tese outcomes suggest that, at a mid-term follow-up, a combination of PRP and HA provides better pain control compared to PRP and, in particular, HA alone.
At three-month follow-up, cases of Group A had higher mean WOMAC scores (36.4) compared to the ones of Group B (19.6) and Group C (21.4). Tese data highlighted a frst difference between cases treated with HA alone and the rest of our cohort, although they were not statistically signifcant to that point due to the limited size of our population (p � 0.076). Tree months later, the mean score of cases treated with HA (Group A) was 31.8, the one of those who received PRP (Group B) was 19.6, and the one of those who received a combination of the two (Group C) was 17.5. Terefore, at this fnal endpoint, those who were injected with PRP and HA had the best functional outcomes according to the WOMAC scoring system. Furthermore, Group C was the only one whose mean value continued to decrease after the frst three months that followed the treatment, suggesting a prolonged efectiveness of the combined approach. Although the diference between Group B and Group C was not sufcient to be considered statistically signifcant according to our endpoints (p > 0.050), the treatment with PRP (Groups B and C together) resulted to be signifcantly better compared to HA alone in terms of clinical outcomes within six months from the end of the treatment.

Discussion
Te combination of HA and PRP represents one of the most promising injective approaches introduced in recent years for moderate knee osteoarthritis, as it could theoretically dovetail the positive characteristics of both of its components. Te hyaluronic acid, a natural component of the synovial fuid, can increase joint viscosity and lubrication but also reduce articular infammation [21]. However, although literature provides evidence that intra-articular injections with HA can ease the symptoms associated with mild-to-moderate osteoarthritis, there is no evidence that the drug could anyhow halt the progression of the disease and these efects tend to decrease months after the treatment [25]. Our results are in line with the ones reported in the literature, as our cases treated with HA injections experienced good results at 3-month control in terms of both pain relief and restoration of knee functionality. However, the same patients did not report further signifcant improvements in the next 3 months. In fact, 6 months after the treatment with HA, patients did not experience any signifcant further pain reduction (according to the visual analogue scale) and they even reported a slight worsening of their knee functionality (according to the WOMAC score).
Platelet-rich plasma, for its part, is another suitable option for today's intra-articular injection treatment in mild-to-moderate knee osteoarthritis. Despite the limited standardization of its production and formulation that limit its introduction in most guidelines, PRP has been shown to reduce pain and infammation associated with knee osteoarthritis. Te literature provides evidence that intra-articular administrations of PRP may infuence tissue regulation due to the high level of platelet-derived growth factors [8,17,[19][20][21]. Our population also confrms PRP's shortterm efectiveness. Comparing the base-time evaluations and the ones at three-month follow-up of all our cohorts, the one treated with PRP had the greatest increase in both VAS and WOMAC scores, thereby suggesting the short-term efectiveness of PRP. Although the WOMAC of those patients remained steady in the following three months and VAS scores even worsened at six-month follow-up, both these scores were higher and had greater improvements from the basal values in comparison with the ones of those cases who received HA injections. In recent years, the dualism between PRP and HA has been partially overcome, as the injection of a combination of the two materials has been proposed and introduced in clinical practice. However, only a limited number of prospective studies have been published so far on this topic [22][23][24][25][26][27]. In 2016, Saturveithan et al. [22] compared the efects of a cycle of PRP + HA with the ones of HA alone in cases sufering from grade III-IV knee osteoarthritis. Te authors reported that those who received PRP alongside HA had much better outcomes in terms of both knee functionality and pain relief within six months after the end of their treatment. Tree years later, in 2019, Papalia et al. [23] published a study with a similar design held on 60 cases sufering from mild-to-moderate knee osteoarthritis (grade II and III according to the Kellgren-Lawrence classifcation). Te authors demonstrated better functional outcomes within 6 months from the treatment for those who received PRP + HA rather than HA alone.
Our study, performed on cases sufering from the same stages of osteoarthritis (grade II and III according to the Kellgren-Lawrence classifcation), did not comprehend only two cohorts that received HA and PRP + HA but also included a third subpopulation that received only PRP, allowing a comparison between the three treatments in a mid-term scenario. In our population, at six-month followup, cases treated with intra-articular injections of PRP + HA had signifcantly lower VAS scores compared to the other cohorts and the absolute lowest WOMAC scores. Furthermore, cases that received the combination of the two treatments were the only ones that reported a remarkable advancement of their functional recovery and pain relief between not only in the frst three months but also between the three and six months after the treatment.
Tese results are in line with the ones already reported in the literature by Lana et al. and Yu et al. [24,25], who carried out studies with comparable designs. However, our study difers from the frst one in terms of knee osteoarthritis grade. Lana et al. evaluated cases with grade I to III according to the Kellgren-Lawrence classifcation, whereas our choice was to exclude cases with incipient knee osteoarthritis, rather focusing on those who were already sufering from mild-to-moderate disease (grade II-III), since they represent the vast majority of those who get the indication for intraarticular injections in our institution. Te paper by Yu et al., for its part, did not specify any radiographic grade of knee osteoarthritis among its inclusion criteria. Tis does not allow a proper comparison between our and their population, despite the convergence of our results. Further randomized controlled studies are going to provide further evidence on the topic in the near future [26].
We are aware that our study is not free of limitations. Although the size of our population (made of 60 cases divided into 3 groups of 20 cases each) is globally comparable to the ones of previous studies on the topic, the lack of greater cohorts represented a restriction for the statistical signifcance of our outcomes. Another limit lies in the duration of our follow-up. Since our endpoint was set to 6 months after the end of injective treatments, we could not extend our horizon any further in order to assess the effectiveness of PRP + HA in a long time period.
Altogether, our prospective study testifes the efectiveness of PRP + HA for cases sufering from grade II and III knee osteoarthritis, as patients' overall outcomes in terms of pain control and functionality resulted to be better than comparable control groups treated with HA and PRP alone in similar conditions. Our experience suggests that a larger use of combined PRP and HA could lead to signifcative benefts for patients with mild-to-moderate knee osteoarthritis. Better pain control and functional performances would translate into a quality of life but also in reduced healthcare spendings. Faced with the reasonable costs of some injections, the treatment with PRP and HA could allow the national health systems to reduce their expenses in terms of painkillers and outpatient visits in the months that follow the treatment.

Conclusions
Our study suggests that the combination of platelet-rich plasma and hyaluronic acid represents an innovative and valuable alternative to the administration of its two components alone. Being capable of reducing both pain and functional impairment within the 6 months that follow its administration, this treatment represents a promising frontier for the nonsurgical approach for patients who sufer from mild-to-moderate knee osteoarthritis.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.